Drosophila presents an ideal model organism for the study of human cis-regulation. Its genome shares the structure and major features of the human genome;all major families of transcription factors, both the basal machinery and site-specific factors;the overall regulatory structure of developmental genes such as the Hox clusters;many of the "master regulatory" transcription factor proteins such as PAX6/eyeless and Distalless that control organ or tissue identity. In many cases, human proteins function in Drosophila just as well as Drosophila proteins. In addition to the shared biology, the fly also offers powerful experimental tools not yet available for mammalian species. Its small genome size, at 130 Mb, is amenable to genome-wide systematic experimentation at a resolution not yet achievable in the human. Additionally, a total of 12 fully sequenced species provide one of the richest comparative genomic datasets, powerful enough to identify individual functional binding sites of regulators across the entire genome. Finally, the wealth of high throughput experimental techniques enables us to validate functional predictions in vivo. In this proposal, we exploit these unique features to produce a comprehensive cis-regulatory map of the Drosophila genome, using a combined experimental and computational approach. We will identify fly promoter regions, short and long range enhancers, insulator and repressor regions, and their defining characteristics based on cis-regulatory motifs and grammars, chromatin state, and transcription factor binding. This project serves as a pilot for the full-scale mapping of functional regulatory elements in the human genome. The methods and strategies developed will be crucial for informing the human genome. In addition, the body of knowledge gained will be invaluable in the understanding of many human diseases and disorders due to regulatory malfunction of gene regulation. Drosophila is an ideal model system for this project, for its compact genome size, experimental resources, and comparative genomics power, while retaining all the major characteristics of the human genome for the study of gene regulation.